Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

Effect of vinpocetine on various types of high-threshold potassium currents in snail neurons

A high-threshold (−20 mV) K⁺ current was recorded from isolated edible snail neurons by a two-microelectrode voltage clamp technique. This current consisted of three components: fast-inactivating K⁺ currents (I_A), noninactivating K⁺ current (I_KD), and Ca²⁺-dependent K⁺ current (I_K(Ca)). Different cells had one to three components of K⁺ current. Vinpocetine increased I_A, moderately inhibited I_KD (by 30–50%) and strongly suppressed I_K(Ca) (by 60–90%). Inhibition of I_K(Ca) was not related to the effect of vinpocetine on the inward Ca²⁺ current. When K⁺ current consisted of all three components, the effect of vinpocetine on the ionic current amplitude was opposite at different potentials. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 10, pp. 408–411, October, 1998     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Fast,persistent, Ca2+-dependent K+ current controls graded electrical activity in crayfish muscle

The early outward current in opener muscle fibres of crayfish (Procambarus clarkii) was studied using the two-electrode voltage-clamp technique. This current was abolished in Ca²⁺-free and 5 mM Cd²⁺ solutions, and was blocked by extra- or intracellular tetraethylammonium, indicating that it was a Ca²⁺-dependent K⁺ current [I _K(Ca)]. I _K(Ca) was voltage dependent, apamin insensitive and sensitive to charybdotoxin (CTX), which, in addition to its tetraethylammonium sensitivity, suggests that the channels mediating I _K(Ca) behave in a BK type manner. I _K(Ca) activation was extremely fast, reaching a maximum within 5 ms, and the inactivation was incomplete, stabilizing at a persistent steady-state. I _K(Ca) was insensitive to intracellular ethylenebis(oxonitrilo)tetraacetate (EGTA), but was abolished by injection of the faster Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA), suggesting that voltage-dependent Ca²⁺ channels and those mediating I _K(Ca) should be clustered closely on the membrane. Under two-electrode current-clamp recording mode, low amplitude, graded responses were evoked under control conditions, whereas repetitive all-or-none spikes were elicited by application of CTX or after loading the cells with BAPTA. We conclude that I _K(Ca) activates extremely quickly, is persistent and is responsible for the generation and control of the low amplitude, graded, active responses of opener muscle fibres.     

Potassium currents in rat colonic smooth muscle cells and changes during development and aging

In a previous study on freshly isolated single smooth muscle cells from the circular layer of the rat distal colon, we reported that the L-type Ca²⁺ current density increased during development and gradually declined with further aging [ZI Xiong, N Sperelakis, N Noffsinger, C Fenoglio-Preiser (1993) Am J Physiol 265: C617–C625]. Since K⁺ current plays a key role in controlling excitability of the cells and hence the motility of the colon, in the present study the voltage-gated K⁺ channel currents, (I _K) were investigated using the whole-cell voltage-clamp technique in colonic myocytes from rats of different ages. A Ca²⁺-sensitive K⁺ current [I _K(Ca)] and two kinds of Ca²⁺-insensitive outward K⁺ currents were identified and characterized. I _K(Ca) was recorded at potentials more positive than –40 mV in Ca²⁺-containing bath solution, and was blocked by Ca²⁺ channel antagonists and tetraethylammonium ion (TEA⁺). After removing Ca²⁺ from the bath solution and using a high ethylenebis(oxonitrilo)tetraacetate (EGTA, 4 mM) concentration in the pipette, two types of Ca²⁺-insensitive I _K were recorded. The first and faster component was usually activated at potentials more positive than –50 mV, and was more sensitive to 4-aminopyridine (4-AP). In contrast, the second and slower (delayed) component was activated at potentials more positive than –30 mV, and was more sensitive to TEA. The total density of the Ca²⁺-insensitive I _K component decreased dramatically during the neonatal period: from 32.2±3.2 pA/pF in 3-day-old rats to 17.8±2.6 pA/pF in 40-day-old rats; there was no further decline during aging (up to 480 days). This total I _K decline was due primarily to a decline in the 4-AP-sensitive component; the TEA-sensitive component remained unchanged at all ages. The possible physiological implications of the decline in density of the 4-AP-sensitive I _K component during neonatal development are discussed.     

Contribution of potassium accumulation in narrow extracellular spaces to the genesis of nicorandil-induced large inward tail current in guinea-pig ventricular cells

The mechanism of nicorandil-induced large inward tail current (I _tail) in single guinea-pig ventricular cells was investigated using the whole-cell patch-clamp technique. In the presence of 0.5–1.0 mM nicorandil, an activator of adenosine 5-triphosphate (ATP)-sensitive K⁺ current (I _KATP), a depolarization pulse causing a large outward current was followed by a large inward I _tail on the repolarization step to the holding potential at-85 mV. The larger the outward current, the greater the I _tail. The amplitude of I _tail increased as a single exponential function (=74.9 ms) as the duration of preceding depolarization was prolonged. Both the outward current and I _tail were inhibited nearly completely after application of glibenclamide (1 M), a specific blocker of I _KATP. Substitution of K⁺ with Cs⁺ in both the external and internal solutions resulted in a virtual elimination of I _tail. I _tail was well preserved under the condition where Ca²⁺ entry during the preceding depolarization was largely inhibited or where external Na⁺ was replaced by Li⁺. A transient positive shift of reversal potential for the net current was observed at the peak of I _tail. At 30 mM external K⁺ concentration, I _tail was almost eliminated. From these findings, it is concluded that the I _tail is a K⁺ current associated with an alteration of the K⁺ equilibrium potential (E _K) following a substantial K⁺ efflux. This E _K change is most likely explained by an accumulation of K⁺in transverse tubules (T-tubules) since I _tail was not induced in atrial cells in which T-tubules are poorly developed.     

Modulation of Ca2+ oscillation and apamin-sensitive,Ca2+-activated K+ current in rat gonadotropes

In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca²⁺ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃ ], which in turn induces an oscillatory activation of apamin-sensitive Ca²⁺-activated K⁺ current, I _K(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) and I _K(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca²⁺]_i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4-phorbol 12,13-didecanoate (4-PDD), reduced the frequency of GnRH-induced [Ca²⁺]_i oscillation and augmented the I _K(Ca) induced by any given level of [Ca²⁺]_i. The slowing of oscillations and the enhancement of I _K(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5-O-(3-thio-triphosphate) (GTP-[S]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P ₃, phorbol esters enhanced I _K(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced I _K(Ca) without affecting [Ca²⁺]_i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca²⁺]_i oscillations probably by actions on phospholipase C, and it enhances I _K(Ca) probably by a direct action on the channels.     

Aromatic aldehydes and aromatic ketones open ATP-sensitive K+ channels in guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of three carbonyl compounds, 3,4-dihydroxy-benzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxy-acetophenone, on the adenosine-5-triphosphate(ATP)-sensitive K⁺ channel current (I _K.ATP) in guinea-pig ventricular myocytes. 3,4-Dihydroxybenzaldehyde (0.5–1 mM) shortened the action potential duration, and this effect was inhibited by application of a specific blocker of I _K.ATP, glibenclamide. The shortening of the action potential duration was shown to be caused by a time-independent outward current. In the cell-attached patch configuration, all three compounds activated a kind of single-channel current, which showed an inward rectification at positive potentials and which had a linear current/voltage relation at negative potentials, having a conductance of 90 pS. The current reversed at about 0 mV in symmetrical K⁺ concentrations on both sides of the membrane. In excised patches this current was blocked by internal application of ATP. Thus we identified this channel as I _K.ATP. The activation effects of two aromatic aldehydes were stronger than that of the aromatic ketone. The effect of these compounds on I _K.ATP was not reduced by addition of cysteine (10 mM). In inside-out patches, 3,4-dihydroxybenzaldehyde increased the activity of I _K.ATP, which had been blocked by 0.5 mM MgATP in the presence of 0.5 mM ADP, but the activation effect was variable and much weaker than that in the cell-attached configuration, and was completely eliminated in the absence of ADP. These results suggest that these compounds: (a) modulate I _K.ATP perhaps through an intracellular mechanism, (b) bind covalently to proteins to form a Schiff base which may by responsible for the effects, and (c) may require an ADP-dependent process.     

The sustained inward current in sino-atrial node cells of guinea-pig heart

 Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current I _f , the delayed rectifier K⁺ current I _K and the L-type Ca²⁺ current I _Ca,L as well as by the absence of both the transient outward current I _to and the inward rectifier K⁺ current I _K,1. After blocking I _f and I _K, depolarizing pulses from –80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca²⁺] ([Ca²⁺]_o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na⁺]_o, differently from I _Ca,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of I _st described in the rabbit SA node pacemaker cells, and that I _st is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither I _f nor I _st. Received: 22 July 1996 / Received after revision: 30 September 1996 / Accepted: 9 October 1996     

Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea

Tracheal smooth muscle cells were enzymatically isolated from guinea-pig trachea. These cells contracted in response to acetylcholine (0.01–10 M) in a concentration-dependent fashion. Under current-clamp conditions with 140 mM K⁺ in the pipette solution, the membrane potential oscillated spontaneously at around –30 mV. Under voltage-clamp conditions, there appeared spontaneous but steady oscillations of outward current (I _o). On depolarization from a holding potential at –40 mV, three components of outward current were elicited: transient outward current (I _T), steady-state outward current (I _s) and I _o. These three components of outward current reversed around the K⁺ equilibrium potential and were abolished by Cs⁺ in the pipette, indicating that K⁺ was the major charge carrier of these outward currents. All these three components were completely suppressed by extracellular tetraethylammonium (10 mM). Both I _T and I _o were depressed by quinidine (1 mM), 4-aminopyridine (10 mM) and nifedipine (100 nM), but I _s was not affected. I _T and I _o were suppressed by a Ca²⁺-free perfusate with less than 1 nM Ca²⁺ in the pipette, while with 10 nM Ca²⁺ in the pipette, only I _o was suppressed. In both conditions, I _s was not affected by the Ca²⁺-free perfusate. Therefore, it is suggested that I _o, I _T and I _s are separate types of K⁺ current. With Cs⁺ in the pipette, K⁺ currents were almost completely suppressed and a transient inward current was observed during depolarizing pulses. The inward current was not affected by tetrodotoxin and increased when the concentration of extracellular Ca²⁺ was raised, indicating that the current is a Ca²⁺ channel current. Even with a holding potential of –80 mV, the low-threshold inward current could not be observed. The high-threshold Ca²⁺ current was abolished by nifedipine (100 nM) and was enhanced by Bay K 8644 (100 nM). The order of permeation of divalent cations through the Ca²⁺ channel was Ba²⁺ >Sr²⁺ Ca²⁺. Cd²⁺ blocked the Ca²⁺ current more effectively than Ni²⁺. These results may indicate that the Ca²⁺ current of tracheal smooth muscle cells is mainly composed of the current through an L-type Ca²⁺ channel.     

Effects of dopamine on ion transport across the rat distal colon

Dopamine (5·10^–6–5·10^–4 M) induced a concentration-dependent decrease in short-circuit current (I_sc) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I_sc. The -adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D₂-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I_sc evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2 Cl^– cotransporter responsible for the uptake of K⁺, and by quinine, a blocker of apical K⁺ channels, indicating that stimulation of K⁺ secretion contributes to the measured change in I_sc. In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K⁺ current. This response was not concomitant with a change in the intracellular [Ca²⁺] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K⁺ secretion.     

Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis

Electrogenic ion transport in the isolated co-Ionic epithelium from normal and transgenic mice with cystic fibrosis (CF mice) has been investigated under short-circuit current (I _sc) conditions. Normal tissues showed chloride secretion in response to carbachol or forskolin, which was sensitive to the Na-K-2Cl cotransport inhibitor, frusemide. Responses to both agents were maintained for at least 12 h in vitro, but the responses to carbachol changed in format throughout this period. By contrast CF colons failed to show the normal secretory responses to carbachol and forskolin, most preparations showing a decrease in I _sc that was immediately reversed by frusemide. In CF colons addition of Ba²⁺ ions or tetraethylammonium (TEA⁺) to the apical bathing solution antagonised the reduction in I _sc caused by the secretagogues. It is concluded that the reduction in I _sc in CF colons is due to electrogenic K⁺ secretion and this was confirmed by flux studies using rubidium-86. In normal colons exposed to TEA⁺ the responses to for-skolin were greater, but not significantly so, presumably because the minor K⁺-secretory responses are dominated by major chloride-secretory responses. Again rubidium-86 fluxes showed an increase of K⁺ secretion in normal colons receiving forskolin. Since the amiloride-sensitive current was not different in CF and normal colons there was no evidence that the CF mice were stressed in a way that increased mineralocorticoid levels and hence K⁺ secretion. Knowledge of the phenotype of the colonic epithelium of the CF mouse sets the baseline from which attempts at gene therapy for the gut must be judged.     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

 Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba²⁺ (holding potential, V _h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca²⁺ channels; T-type Ca²⁺ channels were not seen. By using supramaximal concentrations of selective Ca²⁺ channel blockers, the whole-cell I _Ba could be fractionated into various subcomponents. Thus, I _Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I _Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I _Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca²⁺ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba²⁺. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca²⁺ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca²⁺ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates. Received: 1 May 1998 / Received after revision and accepted: 21 May 1998     

Intracellular Mg2+ inhibits the IP3-activated I K(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording I K(Ca)]

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP₃) can induce an outward Ca²⁺-activated K⁺ current [I _K(Ca)] some neural cells. We have investigated I _K(Ca) activated by intracellular injections of IP₃ in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg²⁺ by citrate. Theoretical calculations suggested free [Mg²⁺] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP₃-activated I _K(Ca) could be recorded when the free Mg²⁺ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg²⁺] blocks I _K(Ca). This appeared to be due to inhibition of the response to released Ca²⁺, since high [Mg²⁺] also blocked the response to intracellular injections of Ca²⁺ ions. Mean Mg²⁺ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP₃-induced I _K(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg²⁺.     

Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents

We sought to determine whether extracellular Ca²⁺ (Ca _e ²⁺ ) and K⁺ (K _e ⁺ ) play essential roles in the normal functioning of cardiac K⁺ channels. Reports by others have shown that removal of Ca _e ²⁺ and K _e ⁺ alters the gating properties of neural delayed rectifier (I _K) and A-type K⁺ currents, resulting in a loss of normal cation selectivity and voltage-dependent gating. We found that removal of Ca _e ²⁺ and K _e ⁺ from the solution bathing guinea pig ventricular myocytes often induced a leak conductance, but did not affect the ionic selectivity or time-dependent activation and deactivation properties of I _K. The effect of [K⁺]_e on the magnitude of the two components of cardiac I _K was also examined. I _K in guinea pig myocytes is comprised of two distinct types of currents: I _Kr (rapidly activating, rectifying) and I _Ks (slowly activating). The differential effect of Ca _e ²⁺ on the two components of I _K (previously shown to shift the voltage dependence of activation of the two currents in opposite directions) was exploited to determine the role of K _e ⁺ on the magnitude of I _Ks and I _Kr. Lowering [K⁺]_e from 4 to 0 mM increased I _Ks, as expected from the change in driving force for K⁺, but decreased I _Kr. The differential effect of [K⁺]_e on the two components of cardiac I _K may explain the reported discrepancies regarding modulation of cardiac I _K conductance by this cation.     

Two types of ω-agatoxin IVA-sensitive Ca2+ channels are coupled to adrenaline and noradrenaline release in bovine adrenal chromaffin cells

 To clarify the role of P-type Ca²⁺ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of ω-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K⁺-evoked depolarization. ω-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component (IC₅₀<1 nM), responsible for 10–15% of catecholamine release evoked by 70 mM K⁺, and a low-potency component that accounted for about 40% of release, with IC₅₀ values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin cells was also inhibited dose dependently by ω-conotoxin MVIIC with IC₅₀ values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM ω-agatoxin IVA and 3 μM ω-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites, presumably, P- and Q-type Ca²⁺ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 ± 4.1%) significantly more than the release of noradrenaline (50 ± 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of K⁺-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show that two types of ω-agatoxin IVA-sensitive Ca²⁺ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion is larger at low levels of depolarization. Received: 6 March 1997 / Received after revision and accepted: 28 April 1997     

Inhibition of apamin-sensitive K+ current by hypoxia in adult rat adrenal chromaffin cells

The effect of hypoxia on small-conductance Ca(2+)-activated K+ current was investigated in a study of adult rat adrenomedullary chromaffin cells (AMCs), which were maintained in short-term culture. The nystatin-perforated, whole-cell patchclamp technique was used to study the effect of hypoxia with minimum perturbation of the intracellular milieu. Under voltage-clamp conditions, acute hypoxia (P(O2) approximately equal to 25 mmHg) suppressed the whole-cell outward currents of more than half the AMCs (24/46). This suppression was eliminated after application of apamin (400 nM), a selective inhibitor of small-conductance Ca(2+)-activated K+ current (I(SK)(Ca)) (n=5), suggesting that an apamin-sensitive component of whole-cell currents is suppressed during hypoxia. In contrast to I(SK)(Ca), Ca2+ current (I(Ca)) (n=10) was not affected by hypoxia. Finally, under current-clamp conditions, hypoxia reversibly depolarized the resting membrane potential of adult AMCs (34/40). Apamin, however, eliminated the hypoxia-induced depolarization (400 nM) (7/8), suggesting that hypoxic depolarization is related to the suppression of I(SK(Ca). From the above results, we conclude that adult AMCs are sensitive to hypoxia, and that I(SK)(Ca) contributes to the hypoxia-induced suppression of whole-cell outward current and depolarization of the resting membrane potential in adult AMCs.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.